Chikungunya (CHIKV), yellow fever (YFV), and Dengue (DENV) are among the most troublesome human health pathogens responsible for hundreds of millions of infections and hundreds of thousands of deaths each year. They are transmitted to human populations by a single mosquito species, Aedes aegypti. Effective human vaccines are not yet available for CHIK, and DENV, and the re-emergence of YFV as a lethal pathogen, in spite of the availability of an effective vaccine, suggests ineffectiveness of vaccination strategies for sustained eradication. Interference with the incubation arboviruses within the arthropod vector is receiving considerable attention as a way to interrupt the viral transmission cycle and reduce the efficiency of transmission to humans. One such approach envisions population replacement of susceptible mosquitoes with those resistant to infection or to halt disease transmission. This approach has distinct advantages of environmentally safety, cost effectiveness, and long-term disease suppression. Transgenic introduction of expressed anti-viral molecules to generate refractoriness in the mosquito vector is now a real possibility. Recent successes reflected in the RIDL approach show significant promise, and can reasonably be expected to be coupled with transgenic refractoriness to establish and maintain transgene effectors in rebound mosquito populations. However, this avenue must necessarily be coupled with ensuring both long term effectiveness and stable transgene expression. Our research is demonstrating that ribozyme (RNA-enzyme) mediated viral suppression can provide an effective means of transgenic immunization against viruses. Recent efforts have confirmed the effectiveness of hammerhead ribozymes and Group I introns in suppressing DENV infection in mosquito cells. The research proposed seeks to build upon our successes in developing anti-viral ribozymes as potent effector genes against arbovirus infections in mosquitoes. In particular, this proposal will focus on developing, optimizing, and validating effective ribozyme suppression strategies against three major emerging and re-emerging disease pathogens vectored by the same mosquito species, Aedes aegypti, DENV, CHIKV, and YF. The ultimate goal is to be able to provide mosquito strains that will be useful in simultaneously eliminating two or more of these viral diseases. PUBLIC HEALTH RELEVANCE: Chikungunya (CHIKV), yellow fever (YFV), and Dengue (DENV) are among the most troublesome single-mosquito transmitted human health pathogens causing hundreds of millions of infections and hundreds of thousands of deaths each year. This proposed research seeks to develop anti-viral ribozymes as means of suppressing the infection of these arbovirus in the mosquito, Aedes aegypti, thereby preventing disease transmission.